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Correlation between choline acetyltransferase activity and learning ability in different mice strains and their offspring
Brain Research, 72 (I 974) 65-70
65
,d;~ Elsevier Scientific Publishing Colnpany, Amsterdam - Printed in The Netherlands
C O R R E L A T I O N BETWEEN C H O L I N E A C E T Y L T R A N S F E R A S E ACTIVITY A N D L E A R N I N G ABILITY IN D I F F E R E N T MICE STRAINS A N D T H E I R OFFSPRING
P. MANDEL, G. AYAD, J. C. HERMETET ANDA. EBEL* Centre de Neurochimie du C.N.R.S., 67085 Strasbourx* Cede.v (France)
(Accepted November 22rid, 1973)
SUMMARY The enzymes related to acetylcholine metabolism were investigated in several cortical regions of 3 inbred strains of mice (SEC, DBA and C57) differing in learning ability, and in their FI offsprings. A higher choline acetyltransferase (ChAc) activity was found in the temporal lobe of mice with high learning ability (SEC and DBA) than in C57 mice which score poorly. The genetic behavioural analysis conducted on the FI offsprings of these strains is demonstrated that FI hybrids of SEC and C57 crosses were able to reach the same high learning levels as the parental SEC strain. In contrast, the DBA >4 C57 offsprings performed as poorly as the C57 strain which had low levels of performance. We showed that ChAc activity in the temporal lobe of the SEC C57 hybrids was close to that of the SEC strain and in the FI offspring of DBA C57 crosses, near the values of the parental line C57. It was suggested that these differences may constitute a neurochemical correlate of the differences in learning abilities of these mice strains.
INTRODUCTION
The prominent role of genetic factors in difference in performance was shown by learning tests conducted on different mouse strains or by the rat selection studies 2,a. ,~,el. Inbred strains of mice which differ in learning ability is may be used as models for investigating biochemical correlates of well defined behavioural patterns such as avoidance, maze learning or wheel running. In a previous report a we discussed metabolism of acetylcholine (ACh) in two * Chargde de recherche au C.N.R.S.
66
I'. MANI)tI_ ~! (d,
mouse strains, DBA/2J characterized by a high level of performance in avoidance and maze learning and C57 BL/6J which showed poor learning abilities 1,e. Cholinergic mechanisms were investigated by measuring choline acetyltransferase (ChAt; f:J.C 2.3. 1.6),the ACh-synthesizing enzyme and its degradation enzyme : acetylcholinesterase (ACHE; EC 3.1.1.7) in several regions of the central nervous system. Our results showed a sharp difference in these enzyme activities in the temporal cortex. DBA mice presented 44~,~i higher activities in the temporal cortex than the (757 strain. It was suggested that these differences may constitute a neurochemical correlate of the differences in learning ability, Actually Penfield has shown that the temporal lobe is involved in memory processes 19,~°. Kliiver and Bucy l°, Horel and Keating s and Mishkin 17 showed that this area is implicated in learning and conditioning processes. Thus, it was of interest to investigate whether differences, similar to those i\)und in DBA and C57 in cholinergic systems, could also be found in other strains which show differences in learning ability as well as in their offspring. When crossing C57 and DBA, the performance of the offspring FI was similar to that of the parent strain C57. On the other hand, when crossing SEC and C57, the parental line SEC seems dominant and the performance of the FI was similar to that of SEC. Thus, an excellent opportunity was available to investigate whether or not the biochemical parameters parallel the learning ability. The enzymes related to acetylcholine metabolism were investigated in different cortical regions of the SEC/I ReJ strain which, like the DBA strain, was able to reach high performance levels. The values obtained are compared with those of the poor performers C57 BL/6J. The same analysis was performed on the FI offspring of SEC and C57 mice and of DBA and C57". METHODS
Male adult mice C57 BL/6J, DBA/2J, SECfl ReJ were provided by the Jackson Laboratory (Bar Harbor, Maine, U.S.A.). The animals were killed by cervical dislocation between 9 and 10 a.m. in order to avoid interference by circadian rhythms. The following zones were used (Fig. I): the frontal cortex anterior to the middle cerebral artery; the parietal cortex posterior to this vessel; the occipital cortex at the posterior hemispheric pole; the temporal lobe at the lower part of the hemisphere, behind the cleft of the middle cerebral artery.
Fig. 1. Dissection of brain zones: 1, cerebellum; 2, occipital cortex; 3, parietal cortex; 4, frontal cortex; 5, olfactory zones: bulb, peduncle and tubereulum; 6, temporal cortex. * A preliminary note concerning DBA and C57 cross offspring has been reported~:L
MICE BRAIN
ChAc ACTIVITY
67
AND LEARNING ABILITY
Limbic structures from the pericallous area, cerebella and the olfactory zones (bulb, peduncle and tuberculum) were also assayed. Immediately after dissection the samples were frozen on dry ice and then homogenized at 0-2 C in 0.5 3/0 Triton X-100 (1 /,J/100 Mg tissue) using Potter-Elvehjem glass homogenizers. Before assay the samples were diluted with 0.05 3{i bovine serum albumin. Choline acetyltransferase was measured according to the microtechnique of McCaman and Hunt 14 as modified by Goldberg et a/. 7, by following the incorporation of [1-14C]acetate from acetyl-CoA into acetylcholine. The [l-HC]acetylcholine formed was precipitated with potassium periodide and the activity of the precipitate counted in a Packard scintillation counter. The enzyme activity was calculated from the known specific activity of the [1-14C]acetyI-CoA (NEN Chemicals, 59.2 mCi/mmole). Acetylcholinesterase was determined by the method of McCaman et al. ta, based on the hydrolysis of [l-14C]acetylcholine (NEN Chemicals, 2.43 mCi/mmole). Unhydrolyzed substrate was precipitated as Reinecke salt. Non-specific cholinesterases were inhibited by addition of iso-octamethyl-pyrophosphoramide (10 ti M). Proteins were measured by the method of Lowry et al. le. RESULTS
The results (Tables l-IV) show that in the parietal, occipital, limbic cortical regions, olfactory bulb and peduncle and cerebellum, no significant differences were found in AChE or ChAc activity between the SEC and C57 strains. In the temporal lobe, the activity of ChAc and AChE was respectively 22 ~,i and ~8 o/ higher in the SEC than in the C57 strain, The differences are highly significant (P 0.002). These results suggest a striking difference in the enzyme activities related TABLE I CHOLINE ACETYLTRANSFERASE ACTIVITY OF DIFFERENT BRAIN RE(lIONS OF S E E
AND
C57 MICE AND OF
THEIR F 1 0 E E S P R I N G
V a l u e s are e x p r e s s e d as ,;;mole/g p r o t e i n / h .
Strain SEC Frontal cortex Temporal cortex kimbiccortex Occipital cortex Parietal cortex Olfactory bulband peduncle Cerebellum
I 1.66" 18.70 11.42 10.18 10.39 29.36 2.02
C57 !: 2.28 ~ 1.72 ~ 1.76 ! 1.28 .... 1.25 : 2.61 ] 0.34
* E a c h v a l u e is the m e a n o f 12 e x p e r i m e n t s ** P 0.002 ( S E C / C 5 7 ) . *** P 0.005 ( C 5 7 / F I ) .
12.24 15.33 10.81 9.60 10.65 25.93 1.90
F] 1.69 0.82** 1.35 1.42 1.78 5.17 0.26
: standard deviation.
12.46 20.33 10.16 9.15 10.55 27.18 1.98
! 1.65 ~ 2.46*** : 1.53 1.57 4:0.11 L 0.46 ~ 0.27
68
P. MANDEL el al.
TABLE 1l ACETYLCHOLINESTERASF ACTIVITY OF DIFFERIF-NT BRAIN REGIONS OF S E C
AND
C57
MI('F AND ()F THEIR
F I OFFSPRING
Values are expressed as mmole/g protein/h. Strain SEC
Frontal cortex 1.06" Temporal cortex 2.14 Limbic cortex 2.52 Occipital cortex 0.97 Parietal cortex 0.96 Olfactory bulb and peduncle 4.56 Cerebellum 0.62
C57
: 0.10 ! 0.20 0.75 ! O. I1 ~ 0.13 0.72 0.07
1.16 1.67 2.43 0.96 0.99 4.71 0.65
FI
± 0.27 __ 0.19"* 5 0.63 ÷ 0.12 ~, 0.06 [1.10 ~: 0.17
1.07 2.09 2.52 0.97 0.96 4.27 0.67
_: 0.17 l:: 0.51"** 0.75 :- 0.1l ~ 0.13 1.36 :!: 0.05
* Each value is the mean of 12 experiments ! standard deviation. ** P < 0.002 (SEC/C57). *** P 0.02 (C57/F1).
t o t h e A C h s y s t e m in t h e t e m p o r a l c o r t e x o f t h e t w o s t r a i n s , as it w a s f o u n d w h e n c o m p a r i n g D B A a n d C575. A s s h o w n i n T a b l e 1, C h A c a c t i v i t y in t h e t e m p o r a l c o r t e x o f t h e F1 o f f s p r i n g o f the SEC x C57 crosses was close to that of the SEC strains. The difference between t h e F1 o f f s p r i n g a n d t h e C 5 7 p a r e n t a l s t r a i n is a b o u t 33 ~ ( P ~ 0.005). T h e d i f f e r e n c e w i t h S E C is n o t s i g n i f i c a n t . A C h E a c t i v i t y ( T a b l e I I ) o f t h e t e m p o r a l l o b e f r o m F I m i c e is a l s o n e a r t h e S E C s t r a i n b u t t h e d i f f e r e n c e w i t h t h e C 5 7 m i c e is n o t s i g n i f i c a n t ( P < 0.2). TABLE Ill THE CHOLINE ACETYLTRANSEERASE ACTIVITY OF DIFFERENT BRAIN R~GIONS OF
DBA AND C57 MICE
AND THEIR F 1 OFFSPRING
Values are expressed as/zmole/g protein/h. Strain DBA
Frontal cortex Temporal cortex Limbic cortex Occipital cortex Parietal cortex Olfactory bulb and peduncle Cerebellum
16.62* 32.09 15.85 13.67 13.37 34.19 2.73
C57
:~ 3.01 ** 7 2.71"** -~: 1A5 - 3.06 ~: 2.55 :! 5.97 t 0.67
11.82 17.72 11.80 11.33 12.83 32.30 2.86
FI
:~ 1.93 ~ 3.52 :!: 3.00 :~ 1.42 :L 4.11 ± 3.74 + 0.56
* Each value is the mean of 16 experiments t- standard deviation. ** P -< 0.01 (DBA/C57). *** P ---: 0,001 (DBA/C57), P .: 0,001 (DBA/FI).
12.87 21.43 11.17 11.09 13.35 3[.89 2.20
1.6t L 1.95 '~ i 1.04 .L: 0.89 2.28 -:_ 3.21 -2- 0.38
MICE BRAIN ChAc
ACllVll
Y AND I.EARNING
69
ABILI] Y
TABLE IV Trig A('ETYLCHOLINI!STERASE ACTIVITY OF DIFFFRFNT BRAIN RE(HONS OF D B A THEIR F I OFFSPRING
ANt)
C57
MI('t! AND
Values are expressed as Itmole/g protein/h. Strain
DBA Frontal cortex 2.15* 0.37 Temporal cortex 3.98 J 0.62** Limbic cortex 3.76 , 0.40 Occipital cortex 2.23 i 0.31 Parietal cortex 2.05 0.55 Olfactory bulb and peduncle 7.72 1.03 Cerebellum 1.72 0.13
('57
1.94 2.26 2.93 1.87 1.88 6.67 1.31
f'l
~ 0.39 ~ 0.49 ~ 0.51 : 0.24 0.37 I 0.97 I 0.15
2.20 3.62 3.21 1.85 1.75 6.32 I.[0
0.30 0.66*** I.OI 0.15 0.12 0.70 0.20
* Each value is the mean of 16 experiments : standard deviation. ** P - 0.001 (DBA/C57). *** P 0.01 (F1~,C57).
In c o n t r a s t C h A c activity (Table 111) in the temporal cortex o f the FI offspring o f the DBA C57 crosses was near that o f the parental line C57. The difference between the F1 oflgpring and DI3A parental strain was a b o u t 33 ,'~,](P 0.001 ). However, A C h E activity (Table IV) o f the t e m p o r a l lobe in FI mice was close to that o f the DBA strain. DISCUSSION
Tire higher enzyme activity in the t e m p o r a l cortex of the SEC and D B A mice may reflect more active regional acetylcholine metabolism in these animals. This observation would present a further s u p p o r t for the previously suggested hypothesis that the cholinergic system o f the t e m p o r a l lobe m a y be involved in learning ability in mice. These observations are in g o o d agreement with other studies showing that both the t e m p o r a l lobe and the cholinergic system are involved in m e m o r y processes 4.s Jo. I;3, 17.19,20
The m o d e o f inheritance o f learning abilities in the 3 strains (SEC, DBA, C57) that we studied biochemically was previously reported by Oliverio et al. ts. The genetic behavioural analysis d e m o n s t r a t e d that F l offsprings o f SEC and C57 cr(~sses were able to reach the same high learning levels in a v o i d a n c e b e h a v i o u r as the parental strain SEC. In contrast, the D B A x C57 offsprings performed as poorly as the parental strain C57. We d e m o n s t r a t e d that C h A c activity in the t e m p o r a l cortex o f these FI offsprings in the cross SEC ~< C57 was close to that o f the SEC strain and in the cross D B A ~ C57 near the values o f the parental line C57. Thus, the genetic correlation oF alterations in the cholinergic system with performance that we had observed in DBA and C57 inbred strains l~ exists also in the SEC strain, and in the FI offsprings o f the different strain crosses, it is relevant to recall that in the c a s e o f t h e S E C . C 5 ? c r o s s e s
70
P. MANDEL el al.
the FI progeny showed a learning ability similar to the high-performance parental line whereas offsprings of the DBA >: C57 strains performed as poorly as the parental strain which had low levels of performance. In both cases the ChAc activity of the t e m p o r a l cortex closely parallels the behaviourat pattern of learning ability, ACKNOWLEDGEMENTS We t h a n k Mr. D. G r u b e r a n d Miss M. Ostertag for skitlfull technical assistance.
REFERENCES 1 BOVET, D., BOVET-NITTI,F., AND OLIVERIO, A., Memory and consolidation mechanisms in avoidance learning of inbred mice, Brain Research, l0 (1968) 168-182. 2 BOVET,D., BOVET-N1TTI,F,, AND OLIVERIO, A., Genetic aspects in learning and memory in mice, Science, 163 (1969) 139-149. 3 BOVET,D., AND OL1VERIO,A., Pharmacogenetic aspects in learning and memory, in preparation. 4 DEUTSCH,J. A., The cholinergic synapse and the site of memory, Science, 174 (1971) 788-794. 5 EBEL, A., HERMETET,J. C., AND MANDEL, P., Comparison of acetylchotinesterase and choline acetyltransferase in temporal cortex of DBA and C57 mice, Nature New Biol., 242 (1973) 56-58, 6 FREDERICSON, E., FINK, C.E., AND PARKER, J.R., Elicitation and inhibition of competitive fighting in food deprived mice, J. genetic Psychol., 86 (1955) 131-141. 7 GOLDBERG,A. M., KAITA,k. A., AND McCAMAN, R. E., Microdetermination of choline acetyltransferase- A comparison of Reinecke vs. periodide precipitation, J. Neurochem.~ 16 (1969) 823-824. 8 HOREL,J. A., AND KEATING,E. G., Partial K1/Jver-Bucy syndrome produced by cortical disconnection, Brain Research, 16 (1969) 281-284. 9 KERKUT,G. A., GEOFFREY,W. O., OLIVER,J. T., RICK, R., AND WALKER, R. J., The effects of drugs on learning in a simple preparation, Comp. gen. Pharmacol., 1 0970) 437-484. 10 KLOVER, H., AND BUCY, P. C., Psychic blindness and other symptoms following bilateral temporal lobectomy in rhesus monkeys, Amer. J. Physiol., ll9 (1937) 352-353. ll LINDZEY,G., LOEHLIN, J., MANOSEVIK,M., AND THIESSEN, D., Behavioral genetics, Ann. Rev. Psychol., 22 (1971) 39-94. 12 LowRY, O. H., ROSEBROUOH,N.J., FARR, A. L., AND RANDALL, R.J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 MANDEL, P., EBEL, A., HERMETET,J. C., BOVET, D., ET OLIVERO, A., Etudes des enzymes du systeme cholinergique chez les hybrides F1 de sonris se distinguant par leur aptitude au conditionnement, C.R. Acad. Sci. (Paris), 276 (1973) 395-398. 14 MCCAMAN,R. E., AND HUNT, J. M., Microdetermination of choline acetylase in nervous tissue, J. Neurochem., 12 (1965) 253-259. 15 McCAMAN, R. W., TOMEY, C. R., AND MCCAMAN, R. E., Radiometric assay of acetylcholinesterase activity in submicrogram amounts of tissue, Life Sci., 7 (1968) 233-244. 16 MCCLEARN,G. E., Behavioral genetics, Ann. Rev. Genet., 4 (I970) 437-468. 17 MISHKIN,M., Visual mechanisms beyond the striate cortex. In R. W. RUSSEL(Ed.), Frontiers in Physiological Psychology, Academic Press, New York, 1966, pp. 93-119. 18 OLIVEmO,A., CASTELLANO,C., AND MESSERI,P., Genetic analysis of avoidance, maze and wheel running behaviors in the mouse, J. comp. Psychol., 79 (1972) 459-473. 19 PENFIELD,W., AND MILNER, B., Memory deficit produced by bilateral lesions in the hippocampal zone, Arch. Neurol. Psychiat. (Chic.), 79 (1958) 475-497. 20 PEROT, P., AND PENFIELD,W., Hallucinations of past experience and experimental responses to stimulus of temporal cortex, Trans. Amer. neurol. Ass., 85 (1960) 80-84. 21 ROYCE,J. R., ANO COVINGTON, M., Genetic differences in the avoidance conditioning in mice, J. comp. physiol. Psychol,, 53 (1960) 197-200.
65
,d;~ Elsevier Scientific Publishing Colnpany, Amsterdam - Printed in The Netherlands
C O R R E L A T I O N BETWEEN C H O L I N E A C E T Y L T R A N S F E R A S E ACTIVITY A N D L E A R N I N G ABILITY IN D I F F E R E N T MICE STRAINS A N D T H E I R OFFSPRING
P. MANDEL, G. AYAD, J. C. HERMETET ANDA. EBEL* Centre de Neurochimie du C.N.R.S., 67085 Strasbourx* Cede.v (France)
(Accepted November 22rid, 1973)
SUMMARY The enzymes related to acetylcholine metabolism were investigated in several cortical regions of 3 inbred strains of mice (SEC, DBA and C57) differing in learning ability, and in their FI offsprings. A higher choline acetyltransferase (ChAc) activity was found in the temporal lobe of mice with high learning ability (SEC and DBA) than in C57 mice which score poorly. The genetic behavioural analysis conducted on the FI offsprings of these strains is demonstrated that FI hybrids of SEC and C57 crosses were able to reach the same high learning levels as the parental SEC strain. In contrast, the DBA >4 C57 offsprings performed as poorly as the C57 strain which had low levels of performance. We showed that ChAc activity in the temporal lobe of the SEC C57 hybrids was close to that of the SEC strain and in the FI offspring of DBA C57 crosses, near the values of the parental line C57. It was suggested that these differences may constitute a neurochemical correlate of the differences in learning abilities of these mice strains.
INTRODUCTION
The prominent role of genetic factors in difference in performance was shown by learning tests conducted on different mouse strains or by the rat selection studies 2,a. ,~,el. Inbred strains of mice which differ in learning ability is may be used as models for investigating biochemical correlates of well defined behavioural patterns such as avoidance, maze learning or wheel running. In a previous report a we discussed metabolism of acetylcholine (ACh) in two * Chargde de recherche au C.N.R.S.
66
I'. MANI)tI_ ~! (d,
mouse strains, DBA/2J characterized by a high level of performance in avoidance and maze learning and C57 BL/6J which showed poor learning abilities 1,e. Cholinergic mechanisms were investigated by measuring choline acetyltransferase (ChAt; f:J.C 2.3. 1.6),the ACh-synthesizing enzyme and its degradation enzyme : acetylcholinesterase (ACHE; EC 3.1.1.7) in several regions of the central nervous system. Our results showed a sharp difference in these enzyme activities in the temporal cortex. DBA mice presented 44~,~i higher activities in the temporal cortex than the (757 strain. It was suggested that these differences may constitute a neurochemical correlate of the differences in learning ability, Actually Penfield has shown that the temporal lobe is involved in memory processes 19,~°. Kliiver and Bucy l°, Horel and Keating s and Mishkin 17 showed that this area is implicated in learning and conditioning processes. Thus, it was of interest to investigate whether differences, similar to those i\)und in DBA and C57 in cholinergic systems, could also be found in other strains which show differences in learning ability as well as in their offspring. When crossing C57 and DBA, the performance of the offspring FI was similar to that of the parent strain C57. On the other hand, when crossing SEC and C57, the parental line SEC seems dominant and the performance of the FI was similar to that of SEC. Thus, an excellent opportunity was available to investigate whether or not the biochemical parameters parallel the learning ability. The enzymes related to acetylcholine metabolism were investigated in different cortical regions of the SEC/I ReJ strain which, like the DBA strain, was able to reach high performance levels. The values obtained are compared with those of the poor performers C57 BL/6J. The same analysis was performed on the FI offspring of SEC and C57 mice and of DBA and C57". METHODS
Male adult mice C57 BL/6J, DBA/2J, SECfl ReJ were provided by the Jackson Laboratory (Bar Harbor, Maine, U.S.A.). The animals were killed by cervical dislocation between 9 and 10 a.m. in order to avoid interference by circadian rhythms. The following zones were used (Fig. I): the frontal cortex anterior to the middle cerebral artery; the parietal cortex posterior to this vessel; the occipital cortex at the posterior hemispheric pole; the temporal lobe at the lower part of the hemisphere, behind the cleft of the middle cerebral artery.
Fig. 1. Dissection of brain zones: 1, cerebellum; 2, occipital cortex; 3, parietal cortex; 4, frontal cortex; 5, olfactory zones: bulb, peduncle and tubereulum; 6, temporal cortex. * A preliminary note concerning DBA and C57 cross offspring has been reported~:L
MICE BRAIN
ChAc ACTIVITY
67
AND LEARNING ABILITY
Limbic structures from the pericallous area, cerebella and the olfactory zones (bulb, peduncle and tuberculum) were also assayed. Immediately after dissection the samples were frozen on dry ice and then homogenized at 0-2 C in 0.5 3/0 Triton X-100 (1 /,J/100 Mg tissue) using Potter-Elvehjem glass homogenizers. Before assay the samples were diluted with 0.05 3{i bovine serum albumin. Choline acetyltransferase was measured according to the microtechnique of McCaman and Hunt 14 as modified by Goldberg et a/. 7, by following the incorporation of [1-14C]acetate from acetyl-CoA into acetylcholine. The [l-HC]acetylcholine formed was precipitated with potassium periodide and the activity of the precipitate counted in a Packard scintillation counter. The enzyme activity was calculated from the known specific activity of the [1-14C]acetyI-CoA (NEN Chemicals, 59.2 mCi/mmole). Acetylcholinesterase was determined by the method of McCaman et al. ta, based on the hydrolysis of [l-14C]acetylcholine (NEN Chemicals, 2.43 mCi/mmole). Unhydrolyzed substrate was precipitated as Reinecke salt. Non-specific cholinesterases were inhibited by addition of iso-octamethyl-pyrophosphoramide (10 ti M). Proteins were measured by the method of Lowry et al. le. RESULTS
The results (Tables l-IV) show that in the parietal, occipital, limbic cortical regions, olfactory bulb and peduncle and cerebellum, no significant differences were found in AChE or ChAc activity between the SEC and C57 strains. In the temporal lobe, the activity of ChAc and AChE was respectively 22 ~,i and ~8 o/ higher in the SEC than in the C57 strain, The differences are highly significant (P 0.002). These results suggest a striking difference in the enzyme activities related TABLE I CHOLINE ACETYLTRANSFERASE ACTIVITY OF DIFFERENT BRAIN RE(lIONS OF S E E
AND
C57 MICE AND OF
THEIR F 1 0 E E S P R I N G
V a l u e s are e x p r e s s e d as ,;;mole/g p r o t e i n / h .
Strain SEC Frontal cortex Temporal cortex kimbiccortex Occipital cortex Parietal cortex Olfactory bulband peduncle Cerebellum
I 1.66" 18.70 11.42 10.18 10.39 29.36 2.02
C57 !: 2.28 ~ 1.72 ~ 1.76 ! 1.28 .... 1.25 : 2.61 ] 0.34
* E a c h v a l u e is the m e a n o f 12 e x p e r i m e n t s ** P 0.002 ( S E C / C 5 7 ) . *** P 0.005 ( C 5 7 / F I ) .
12.24 15.33 10.81 9.60 10.65 25.93 1.90
F] 1.69 0.82** 1.35 1.42 1.78 5.17 0.26
: standard deviation.
12.46 20.33 10.16 9.15 10.55 27.18 1.98
! 1.65 ~ 2.46*** : 1.53 1.57 4:0.11 L 0.46 ~ 0.27
68
P. MANDEL el al.
TABLE 1l ACETYLCHOLINESTERASF ACTIVITY OF DIFFERIF-NT BRAIN REGIONS OF S E C
AND
C57
MI('F AND ()F THEIR
F I OFFSPRING
Values are expressed as mmole/g protein/h. Strain SEC
Frontal cortex 1.06" Temporal cortex 2.14 Limbic cortex 2.52 Occipital cortex 0.97 Parietal cortex 0.96 Olfactory bulb and peduncle 4.56 Cerebellum 0.62
C57
: 0.10 ! 0.20 0.75 ! O. I1 ~ 0.13 0.72 0.07
1.16 1.67 2.43 0.96 0.99 4.71 0.65
FI
± 0.27 __ 0.19"* 5 0.63 ÷ 0.12 ~, 0.06 [1.10 ~: 0.17
1.07 2.09 2.52 0.97 0.96 4.27 0.67
_: 0.17 l:: 0.51"** 0.75 :- 0.1l ~ 0.13 1.36 :!: 0.05
* Each value is the mean of 12 experiments ! standard deviation. ** P < 0.002 (SEC/C57). *** P 0.02 (C57/F1).
t o t h e A C h s y s t e m in t h e t e m p o r a l c o r t e x o f t h e t w o s t r a i n s , as it w a s f o u n d w h e n c o m p a r i n g D B A a n d C575. A s s h o w n i n T a b l e 1, C h A c a c t i v i t y in t h e t e m p o r a l c o r t e x o f t h e F1 o f f s p r i n g o f the SEC x C57 crosses was close to that of the SEC strains. The difference between t h e F1 o f f s p r i n g a n d t h e C 5 7 p a r e n t a l s t r a i n is a b o u t 33 ~ ( P ~ 0.005). T h e d i f f e r e n c e w i t h S E C is n o t s i g n i f i c a n t . A C h E a c t i v i t y ( T a b l e I I ) o f t h e t e m p o r a l l o b e f r o m F I m i c e is a l s o n e a r t h e S E C s t r a i n b u t t h e d i f f e r e n c e w i t h t h e C 5 7 m i c e is n o t s i g n i f i c a n t ( P < 0.2). TABLE Ill THE CHOLINE ACETYLTRANSEERASE ACTIVITY OF DIFFERENT BRAIN R~GIONS OF
DBA AND C57 MICE
AND THEIR F 1 OFFSPRING
Values are expressed as/zmole/g protein/h. Strain DBA
Frontal cortex Temporal cortex Limbic cortex Occipital cortex Parietal cortex Olfactory bulb and peduncle Cerebellum
16.62* 32.09 15.85 13.67 13.37 34.19 2.73
C57
:~ 3.01 ** 7 2.71"** -~: 1A5 - 3.06 ~: 2.55 :! 5.97 t 0.67
11.82 17.72 11.80 11.33 12.83 32.30 2.86
FI
:~ 1.93 ~ 3.52 :!: 3.00 :~ 1.42 :L 4.11 ± 3.74 + 0.56
* Each value is the mean of 16 experiments t- standard deviation. ** P -< 0.01 (DBA/C57). *** P ---: 0,001 (DBA/C57), P .: 0,001 (DBA/FI).
12.87 21.43 11.17 11.09 13.35 3[.89 2.20
1.6t L 1.95 '~ i 1.04 .L: 0.89 2.28 -:_ 3.21 -2- 0.38
MICE BRAIN ChAc
ACllVll
Y AND I.EARNING
69
ABILI] Y
TABLE IV Trig A('ETYLCHOLINI!STERASE ACTIVITY OF DIFFFRFNT BRAIN RE(HONS OF D B A THEIR F I OFFSPRING
ANt)
C57
MI('t! AND
Values are expressed as Itmole/g protein/h. Strain
DBA Frontal cortex 2.15* 0.37 Temporal cortex 3.98 J 0.62** Limbic cortex 3.76 , 0.40 Occipital cortex 2.23 i 0.31 Parietal cortex 2.05 0.55 Olfactory bulb and peduncle 7.72 1.03 Cerebellum 1.72 0.13
('57
1.94 2.26 2.93 1.87 1.88 6.67 1.31
f'l
~ 0.39 ~ 0.49 ~ 0.51 : 0.24 0.37 I 0.97 I 0.15
2.20 3.62 3.21 1.85 1.75 6.32 I.[0
0.30 0.66*** I.OI 0.15 0.12 0.70 0.20
* Each value is the mean of 16 experiments : standard deviation. ** P - 0.001 (DBA/C57). *** P 0.01 (F1~,C57).
In c o n t r a s t C h A c activity (Table 111) in the temporal cortex o f the FI offspring o f the DBA C57 crosses was near that o f the parental line C57. The difference between the F1 oflgpring and DI3A parental strain was a b o u t 33 ,'~,](P 0.001 ). However, A C h E activity (Table IV) o f the t e m p o r a l lobe in FI mice was close to that o f the DBA strain. DISCUSSION
Tire higher enzyme activity in the t e m p o r a l cortex of the SEC and D B A mice may reflect more active regional acetylcholine metabolism in these animals. This observation would present a further s u p p o r t for the previously suggested hypothesis that the cholinergic system o f the t e m p o r a l lobe m a y be involved in learning ability in mice. These observations are in g o o d agreement with other studies showing that both the t e m p o r a l lobe and the cholinergic system are involved in m e m o r y processes 4.s Jo. I;3, 17.19,20
The m o d e o f inheritance o f learning abilities in the 3 strains (SEC, DBA, C57) that we studied biochemically was previously reported by Oliverio et al. ts. The genetic behavioural analysis d e m o n s t r a t e d that F l offsprings o f SEC and C57 cr(~sses were able to reach the same high learning levels in a v o i d a n c e b e h a v i o u r as the parental strain SEC. In contrast, the D B A x C57 offsprings performed as poorly as the parental strain C57. We d e m o n s t r a t e d that C h A c activity in the t e m p o r a l cortex o f these FI offsprings in the cross SEC ~< C57 was close to that o f the SEC strain and in the cross D B A ~ C57 near the values o f the parental line C57. Thus, the genetic correlation oF alterations in the cholinergic system with performance that we had observed in DBA and C57 inbred strains l~ exists also in the SEC strain, and in the FI offsprings o f the different strain crosses, it is relevant to recall that in the c a s e o f t h e S E C . C 5 ? c r o s s e s
70
P. MANDEL el al.
the FI progeny showed a learning ability similar to the high-performance parental line whereas offsprings of the DBA >: C57 strains performed as poorly as the parental strain which had low levels of performance. In both cases the ChAc activity of the t e m p o r a l cortex closely parallels the behaviourat pattern of learning ability, ACKNOWLEDGEMENTS We t h a n k Mr. D. G r u b e r a n d Miss M. Ostertag for skitlfull technical assistance.
REFERENCES 1 BOVET, D., BOVET-NITTI,F., AND OLIVERIO, A., Memory and consolidation mechanisms in avoidance learning of inbred mice, Brain Research, l0 (1968) 168-182. 2 BOVET,D., BOVET-N1TTI,F,, AND OLIVERIO, A., Genetic aspects in learning and memory in mice, Science, 163 (1969) 139-149. 3 BOVET,D., AND OL1VERIO,A., Pharmacogenetic aspects in learning and memory, in preparation. 4 DEUTSCH,J. A., The cholinergic synapse and the site of memory, Science, 174 (1971) 788-794. 5 EBEL, A., HERMETET,J. C., AND MANDEL, P., Comparison of acetylchotinesterase and choline acetyltransferase in temporal cortex of DBA and C57 mice, Nature New Biol., 242 (1973) 56-58, 6 FREDERICSON, E., FINK, C.E., AND PARKER, J.R., Elicitation and inhibition of competitive fighting in food deprived mice, J. genetic Psychol., 86 (1955) 131-141. 7 GOLDBERG,A. M., KAITA,k. A., AND McCAMAN, R. E., Microdetermination of choline acetyltransferase- A comparison of Reinecke vs. periodide precipitation, J. Neurochem.~ 16 (1969) 823-824. 8 HOREL,J. A., AND KEATING,E. G., Partial K1/Jver-Bucy syndrome produced by cortical disconnection, Brain Research, 16 (1969) 281-284. 9 KERKUT,G. A., GEOFFREY,W. O., OLIVER,J. T., RICK, R., AND WALKER, R. J., The effects of drugs on learning in a simple preparation, Comp. gen. Pharmacol., 1 0970) 437-484. 10 KLOVER, H., AND BUCY, P. C., Psychic blindness and other symptoms following bilateral temporal lobectomy in rhesus monkeys, Amer. J. Physiol., ll9 (1937) 352-353. ll LINDZEY,G., LOEHLIN, J., MANOSEVIK,M., AND THIESSEN, D., Behavioral genetics, Ann. Rev. Psychol., 22 (1971) 39-94. 12 LowRY, O. H., ROSEBROUOH,N.J., FARR, A. L., AND RANDALL, R.J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 MANDEL, P., EBEL, A., HERMETET,J. C., BOVET, D., ET OLIVERO, A., Etudes des enzymes du systeme cholinergique chez les hybrides F1 de sonris se distinguant par leur aptitude au conditionnement, C.R. Acad. Sci. (Paris), 276 (1973) 395-398. 14 MCCAMAN,R. E., AND HUNT, J. M., Microdetermination of choline acetylase in nervous tissue, J. Neurochem., 12 (1965) 253-259. 15 McCAMAN, R. W., TOMEY, C. R., AND MCCAMAN, R. E., Radiometric assay of acetylcholinesterase activity in submicrogram amounts of tissue, Life Sci., 7 (1968) 233-244. 16 MCCLEARN,G. E., Behavioral genetics, Ann. Rev. Genet., 4 (I970) 437-468. 17 MISHKIN,M., Visual mechanisms beyond the striate cortex. In R. W. RUSSEL(Ed.), Frontiers in Physiological Psychology, Academic Press, New York, 1966, pp. 93-119. 18 OLIVEmO,A., CASTELLANO,C., AND MESSERI,P., Genetic analysis of avoidance, maze and wheel running behaviors in the mouse, J. comp. Psychol., 79 (1972) 459-473. 19 PENFIELD,W., AND MILNER, B., Memory deficit produced by bilateral lesions in the hippocampal zone, Arch. Neurol. Psychiat. (Chic.), 79 (1958) 475-497. 20 PEROT, P., AND PENFIELD,W., Hallucinations of past experience and experimental responses to stimulus of temporal cortex, Trans. Amer. neurol. Ass., 85 (1960) 80-84. 21 ROYCE,J. R., ANO COVINGTON, M., Genetic differences in the avoidance conditioning in mice, J. comp. physiol. Psychol,, 53 (1960) 197-200.